Assessment of adaptive focused acoustics versus manual vortex/freeze-thaw for intracellular metabolite extraction from Streptomyces lividans producing recombinant proteins using GC-MS and multi-block principal component analysis.
نویسندگان
چکیده
We compared the gas chromatography-mass spectrometry (GC-MS) metabolite profiles of mouse tumour necrosis factor alpha (mTNF-alpha) secreting Streptomyces lividans TK24 to the non-secreting wild type and the wild type harbouring the empty pIJ486 plasmid by multi-block principal component analysis (PCA). The multi-block PCA model successfully identified peaks that were statistically different between the protein secreting and non-secreting strains, and at the same time also uncovered the efficiency of intracellular metabolite extraction by an ultrasonic adaptive focused acoustics (AFA) technique compared to a manual vortex/freeze-thaw method. Fifty-one metabolites were significantly different between the three biological strains and 17 of these were abundant in the mTNF-alpha secreting strain compared to the non-secreting strains. No significant differences in the number of detected metabolite peaks were observed between the two extraction techniques. However, from the loadings of the multi-block PCA model, as well as univariate statistical analysis, we observed that the relative peak response ratios to the internal standard of 10 metabolites were higher for the AFA extraction, suggesting a more efficient recovery of these metabolites than achieved with the manual vortex/freeze thaw method.
منابع مشابه
Metabolomics investigation of recombinant mTNFα production in Streptomyces lividans
BACKGROUND Whilst undergoing differentiation, Streptomyces produce a large quantity of hydrolytic enzymes and secondary metabolites, and it is this very ability that has focussed increasing interest on the use of these bacteria as hosts for the production of various heterologous proteins. However, within this genus, the exploration and understanding of the metabolic burden associated with such ...
متن کاملGlobal metabolic profiling of Escherichia coli cultures: an evaluation of methods for quenching and extraction of intracellular metabolites.
Metabolomics and systems biology require the acquisition of reproducible, robust, reliable, and homogeneous biological data sets. Therefore, we developed and validated standard operating procedures (SOPs) for quenching and efficient extraction of metabolites from Escherichia coli to determine the best methods to approach global analysis of the metabolome. E. coli was grown in chemostat culture ...
متن کاملMaking It Last: Storage Time and Temperature Have Differential Impacts on Metabolite Profiles of Airway Samples from Cystic Fibrosis Patients
Metabolites of human or microbial origin have the potential to be important biomarkers of the disease state in cystic fibrosis (CF). Clinical sample collection and storage conditions may impact metabolite abundances with clinical relevance. We measured the change in metabolite composition based on untargeted gas chromatography-mass spectrometry (GC-MS) when CF sputum samples were stored at 4°C,...
متن کاملPhytochemical Profiles and Antimicrobial Activities of Allium cepa Red cv. and A. sativum Subjected to Different Drying Methods: A Comparative MS-Based Metabolomics.
Plants of the Allium genus produce sulphur compounds that give them a characteristic (alliaceous) flavour and mediate for their medicinal use. In this study, the chemical composition and antimicrobial properties of Allium cepa red cv. and A. sativum in the context of three different drying processes were assessed using metabolomics. Bulbs were dried using either microwave, air drying, or freeze...
متن کاملTesting extraction and storage parameters for a fecal hormone method.
Four experiments were conducted to test different aspects of a "field-friendly" fecal hormone extraction method that utilizes methanol extraction in the field followed by storage on C18 solid-phase extraction cartridges. Fecal samples were collected from geladas (Theropithecus gelada) housed at the Bronx Zoo, and the experiments were conducted in a laboratory setting to ensure maximum control. ...
متن کاملذخیره در منابع من
با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید
برای دانلود متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید
ثبت ناماگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید
ورودعنوان ژورنال:
- The Analyst
دوره 135 5 شماره
صفحات -
تاریخ انتشار 2010